Transient isotachophoresis-Capillary zone electrophoresis-Mass spectrometry method with off-line microscale solid phase extraction pretreatment for quantitation of intact low molecular mass proteins in various biological fluids

Abstract

Quantification of proteins is still predominantly done by the traditional bottom-up approach. Targeting of intact proteins in complex biological matrices is connected with multiple challenges during the sample pretreatment, separation, and detection step of the analytical workflow. In this work, we focused on the development of an on-line hyphenated capillary zone electrophoresis-mass spectrometry method employing off-line microscale solid-phase extraction based on hydrophilic lipophilic balance (HLB) sorbent as a sample pretreatment step for the analysis of low molecular mass intact proteins (<20 kDa) spiked in various biological fluids (human serum, plasma, urine, and saliva). A detailed optimization process involved the selection of a suitable capillary surface, background electrolyte (BGE), and comparison of two in-capillary preconcentration methods, namely transient isotachophoresis (tITP) and dynamic pH junction (DPJ), to enhance the sensitivity of the method. Optimum separation of the analytes was achieved using uncoated bare fused silica capillary employing 500 mM formic acid (pH 1.96) + 5 % (v/v) acetonitrile as BGE. tITP was utilized as an optimum preconcentration technique, achieving a 19- to 127-fold increase in the signal intensity when using 200 mM ammonium formate (adjusted to pH 4.00) as the leading electrolyte and BGE as the terminating electrolyte. Off-line microscale solid-phase extraction with various eluate treatment procedures was evaluated to ensure the compatibility of the sample pretreatment method with the selected in-capillary preconcentration, separation, and detection process. Achieved extraction recoveries of spiked proteins were in the range of 76-100 % for urine, 12-54 % for serum, 21-106 % for plasma, and 25-98 % for saliva when the eluate was evaporated and reconstituted in the solution of the leading electrolyte to achieve the tITP process. The optimum method was validated across different biological matrices, offering good linearity, accuracy, and precision, and making it suitable for proteomic studies (e.g., therapeutic drug monitoring, biomarker research) in different biological samples.

Keywords: Biological matrix; Capillary electrophoresis; Intact proteins; Preconcentration; Sample preparation.